This invention relates to reversed-phase materials and methods for producing the same and more particularly relates to hydrophobic reversed-phase materials that are particularly well suited as packing materials for reversed-phase chromatography.
High performance reversed-phase chromatography has become a powerful tool for the isolation of natural and synthetic polypeptides. With the development of efficient wide pore macroparticulate media and mobile phases capable of dissolving large denatured polypeptides, peptide fragments can be isolated in sufficient purity for sequence analysis.
The stationary phases most commonly used in silica-based reversed-phase chromatography columns are straight chain alkanes of 4 to 18 carbon atoms. These alkyl ligands are covalently coupled to silanol groups on the silica surface by reaction with an alkylchlorosilane to form a siloxane bond. However, due to steric limitations, the addition of bulky alkyl silane groups at the silica surface is not quantitative and a number of free silanols remain. These residual hydroxyl moieties may be partially sequestered in a second end capping reaction with chloromethylsilanes.
Recent work has shown that in the reversed-phase chromatography of some membrane proteins, the addition of 40 to 60% formic acid to the mobile phase is required to solubilize these very hydrophobic polypeptides. The use of strongly acidic mobile phases presents a problem with the current generation of silica-based reversed-phase chromatography columns. Since the Si-C bond is more susceptible to attack by electrophiles than a C-C bond, strong acids, e.g. formic acid, can gradually cleave the Si-C bond between the silica support and the stationary phase.
Previous work by Alpert and Regnier with adsorbed polyethyleneimine chemistry as discussed in U.S. Pat. No. 4,245,005, the teachings of which are incorporated herein by reference, has shown it to be extremely versatile for the synthesis of anion-exchange stationary phases. Utilizing the existing adsorption technology pioneered by Alpert and Regnier for the synthesis of adsorbed polymeric anion-exchange media, reversed-phase materials of the invention have been produced.